RNA oligonucleotides were end-labelled with [γ32P]-ATP using Polynucleotide Kinase (Fermentas). UV cross-linking with proteins was performed as previously described [10] (link). For the in vitro reconstitution assay, 10 or 100 µg ORF578–120 was incubated with 5 µg radiolabelled and cold RNA (7merS or 14merS) at room temperature for 10 minutes. The mixture was added to 20 µg of GST-tagged full length ALYREF (aa1–218) immobilized onto Glutathione-coated beads (GE Healthcare) in RB100 buffer. Beads were washed and complexes were eluted in native conditions (50 mM TRIS pH 7.5, 100 mM NaCl, 40 mM reduced glutathione) before being subjected to UV-irradiation or not. Proteins were resolved on 15% SDS-PAGE stained with Coomassie blue and analyzed by PhosphoImaging.
Polynucleotide kinase
Manufactured by Thermo Fisher Scientific
Sourced in Germany, India
Polynucleotide kinase is an enzyme that catalyzes the transfer of a phosphate group from ATP to the 5' hydroxyl terminus of DNA, RNA, or other polynucleotides. It is a key enzyme used in various molecular biology techniques, such as DNA ligation and end-labeling of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (2 mentions)
γ 32p atp, by Hartmann Analytic (2 mentions)
Phosphorimager, by GE Healthcare (2 mentions)
γ 32p atp, by PerkinElmer (2 mentions)
Glutathione coated beads, by GE Healthcare (1 mentions)
Microspin g 50 column, by GE Healthcare (1 mentions)
Megascript high yield transcription kit, by Thermo Fisher Scientific (1 mentions)
Calf intestine alkaline phosphatase, by New England Biolabs (1 mentions)
Phosphor screen, by GE Healthcare (1 mentions)
Rna loading dye, by Thermo Fisher Scientific (1 mentions)
Gotaq dna polymerase, by Promega (1 mentions)
Qiaprep miniprep kit, by Qiagen (1 mentions)
Rapid dna dephos ligation kit, by Roche (1 mentions)
Oligonucleotides, by Metabion (1 mentions)
Probequant g 50 micro column, by GE Healthcare (1 mentions)
Typhoon 9200 phosphorimager, by GE Healthcare (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by Thermo Fisher Scientific (1 mentions)
Restriction endonuclease, by Thermo Fisher Scientific (1 mentions)
27 protocols using polynucleotide kinase
1
In vitro Reconstitution of ALYREF Binding
2
In Vitro Transcript Synthesis and Labeling
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Transcripts used for in vitro assays (EMSA, RNA structure probing, 30S ribosome toeprinting) were synthesized with MEGAscript High Yield Transcription Kit (AM1333, Ambion). DNA templates with T7 promoter sequences were generated by PCR with oligonucleotides listed in Appendix Table S2 . RNA was isolated with phenol:chloroform:isopropanol (25:24:1), ethanol‐precipitated at −80°C and gel‐purified. For labelling, 20 pmol RNA was dephosphorylated with 10 units of calf intestine alkaline phosphatase (New England Biolabs) in a 20 μl reaction at 37°C for 1 h, followed by purification and precipitation, as above. The dephosphorylated RNA was 5′‐labelled with 3 μl of 32P‐γ‐ATP (10 Ci/l, 3,000 Ci/mmol) and 1 unit of polynucleotide kinase (Fermentas) for 1 h at 37°C in a 20 μl reaction. Unincorporated nucleotides were removed with Microspin G‐50 Columns (GE Healthcare), followed by purification of the labelled RNA on a denaturing polyacrylamide gel. Upon visualization of the labelled RNA with phosphorimager, the RNA band was excised from the gel and eluted with 0.1 M sodium acetate, 0.1% SDS, 10 mM EDTA at 4°C overnight, followed by phenol extraction and precipitation as before. Final concentrations were checked by NanoDrop 2000.
3
Quantitative Crosslinking Assay for siRNA
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The substrate oligonucleotides were radioactively labeled using polynucleotide kinase (Fermentas) and γ32P-ATP; the non-labeled strands were phosphorylated with non-radioactive ATP. 10nM radioactively labeled, thiouridine containing siRNA duplexes were incubated with 1 μM dsRBD1, 1 μM dsRBD2, 500 nM LoqsPDΔNC, 500 nM LoqsPD or 250 nM Dcr-2/LoqsPD complex for 30 min at room temperature. Crosslinking was induced with 3 × 500 mJ/cm2 of 365 nm wavelength light, and the samples were resolved on a 10 or 15% PAA gel for 1 h at 170 V. The radioactive signals were read out using a phosphorimager screen on a Typhoon imager and quantified using Multigauge V3.0 software.
4
Comparative RNase E Cleavage of Polyadenine
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20-mer polyadenine (A20), polyadenine with a uracil residue at position 15 (A20U), and polyadenine with a pseudouridine residue at position 15 (A20ψ) were obtained from Dharmacon. Oligoribonucleotides were 5′ labeled with 32P using polynucleotide kinase (Fermentas), according to manufacturer instructions. Assays were carried out in reaction buffer (25 mM Tris-HCl pH 7.5, 50 mM NaCl, 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.5 U/µL RNase OUT) at 37°C. 100 nM purified RNase E NTD was used for the reactions. Time course reactions were stopped at indicated time points by addition of STOP solution (20 mM EDTA, 2% w/v SDS). RNA loading dye (Thermo Fisher) was added to samples which were denatured (98°C, 2 min) and loaded onto polyacrylamide gels containing 7.5 M urea. Gels were dried and exposed to phosphor screens (GE Healthcare) and the signal analyzed with TyphoonT 9400 (GE Healthcare).
5
Plasmid Isolation and Transformation
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Plasmid isolation, molecular cloning and transformation of E. coli as well as electrophoresis were performed using standard procedures [61 ]. Transformation of C. glutamicum was performed by electroporation as described previously [62 (link)]. Chromosomal DNA of C. glutamicum was prepared as formerly described [63 (link)]. PCR experiments were performed using GoTaq DNA polymerase (Promega) or KOD Hot Start polymerase (Novagen) with oligonucleotides obtained from Metabion (listed in Additional file 1 : Table S4). All restriction enzymes and polynucleotide kinase were obtained from Fermentas and used according to the manufacturer’s instructions. Dephosphorylation and ligation were performed using the Rapid DNA Dephos & Ligation Kit from Roche. Plasmids were isolated from E. coli using the QIAprep miniprep kit (Qiagen, Hilden, Germany). DNA sequencing was used to control all inserts of the plasmid constructs listed in Table 1 .
6
Protein-DNA Binding Assay for AP1 Sites
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Double-stranded oligos (sequences listed in S2 Table ) were labelled with 32P (GE Healthcare) using Polynucleotide Kinase (Fermentas) and purified with illustra ProbeQuant G-50 Micro columns (GE Healthcare). 5–10 fmol/μl were used in binding reactions with 50 ng of recombinant protein in Bclw/BSA buffer (10% Glycerol, 10 mM Hepes pH 7.9, 70 mM NaCl, 0.2 mM EDTA, 4 mM MgCl2, 25 mM DTT, 50 μM Zn, 0.1 mg/ml BSA and 50 ng/μl polydI-dC). Binding reactions were performed for 30–40 min at 30°C and separated on a 4% polyacrylamide gel with 0.25x TBE (89 mM Tris, 89 mM boric acid, and 1 mM EDTA) as running buffer for 1h. The gel was dried, exposed and then visualized by using Typhoon 9200 PhosphorImager (GE Healthcare). For oligo competitions, a 100-fold molar excess of unlabeled DNA fragments containing either wild-type or mutated distal AP1 sequences was included at the beginning of the reaction.
7
Cloning and Protein Expression in E. coli
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DH5α and BL21(DE3)pLysS strains of E. coli are maintained as laboratory stocks. Plasmid pYUB28b and strain Mycobacterium smegmatis mc2 4517 were a kind gift from Dr. Ghader Bashiri, New Zealand [18 ]. Oligonucleotide primers were synthesized on order, in desalted form, by Sigma–Aldrich Chemicals Pvt. Ltd., India.
Restriction endonucleases, T4 DNA ligase, DNA molecular weight marker, dNTPs, Pfu DNA polymerase, polynucleotide kinase, DNA ligase and Taq DNA polymerase were procured from MBI Fermentas, Germany. Protein molecular weight markers were procured from Bangalore Genei or Sigma Aldrich Chemicals Pvt. Ltd., India. Agarose, calcium chloride, PMSF, DTT, acrylamide, N,N′-methylene-acrylamide, Coomassie brilliant blue-G and -R, APS, TEMED were procured from Sigma-Aldrich Chemicals Pvt. Ltd., India. Thrombin was purchased from Novagen, USA. Streptococcus thermophilus UDP-Gal 4-epimerase was purchased from Calbiochem, USA. Antibiotics and IPTG were from MP Biomedicals, India or Himedia, India. Culture media were from Himedia, India. Qiaexpress Ni-NTA spin kits were procured from Qiagen, USA. All other chemicals were of analytical reagent grade procured from Sisco Research Laboratories, India or Himedia, India. Protease inhibitor cocktail tablets were purchased from Sigma Aldrich Chemicals Pvt. Ltd., India.
Restriction endonucleases, T4 DNA ligase, DNA molecular weight marker, dNTPs, Pfu DNA polymerase, polynucleotide kinase, DNA ligase and Taq DNA polymerase were procured from MBI Fermentas, Germany. Protein molecular weight markers were procured from Bangalore Genei or Sigma Aldrich Chemicals Pvt. Ltd., India. Agarose, calcium chloride, PMSF, DTT, acrylamide, N,N′-methylene-acrylamide, Coomassie brilliant blue-G and -R, APS, TEMED were procured from Sigma-Aldrich Chemicals Pvt. Ltd., India. Thrombin was purchased from Novagen, USA. Streptococcus thermophilus UDP-Gal 4-epimerase was purchased from Calbiochem, USA. Antibiotics and IPTG were from MP Biomedicals, India or Himedia, India. Culture media were from Himedia, India. Qiaexpress Ni-NTA spin kits were procured from Qiagen, USA. All other chemicals were of analytical reagent grade procured from Sisco Research Laboratories, India or Himedia, India. Protease inhibitor cocktail tablets were purchased from Sigma Aldrich Chemicals Pvt. Ltd., India.
8
Transcriptome Library Construction and Sequencing
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Transcriptome libraries were constructed using Ion Total RNA-Seq Kit v2 (Life Technologies) [60 ] with the following modifications of the protocol. For RNA fragmentation 1 ul of 10x RNase III buffer (Life Technologies) was added to 9 ul of RNA solution and heated for 10 min at 95°C followed by immediate snap-cooling on ice. After that 1 ul of 10 uM ATP and 1 ul of polynucleotide kinase (Fermentas) were added to the solution from the previous step and the whole mix was incubated at 37°C for 30 min. Fragmented RNA was cleaned up using Micro Bio-Spin Chromatography Columns (Bio-Rad). Further steps of library preparation including adapter ligation, first-strand cDNA synthesis and amplification were carried out in accordance with manufacturer's instructions. The prepared library was purified by magnetic beads Agencourt AMPure XP (Beckman Coulter Inc) and its quality was assessed by 2100 Bioanalyzer (Agilent Genomics) using Agilent High Sensitivity DNA Kit (Agilent Genomics). The sequencing of constructed transcriptome libraries was performed on Ion Proton platform using ION PI HI-Q Sequencing 200 Kit and Ion PI Chip Kit v2 (Life Technologies) following the recommendations of the manufacturer.
9
Hfq Protein-RNA Interaction Assay
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The antR(-95–+67) and PrrF2 RNAs were de-phosphorylated with FastAP thermo sensitive alkaline phosphatase (Thermo Scientific), and subsequently 5´-end labeled using [γ-32P]-ATP (Hartmann Analytic) and polynucleotide kinase (Thermo Scientific). The labelled RNAs were gel-purified following standard procedures, eluted and kept in diethylpyrocarbonate- (DEPC) treated water. Labeled RNA (0.1 pmol) dissolved in DEPC water was incubated with increasing amounts of HfqPae, HfqPaeY25DFlag and HfqPaeK56A proteins (S3 Fig ) or unlabeled RNA and Hfq (Figs 1C and S5 ) in 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 60 mM NaCl, 10 mM NaH2PO4, 10 mM DTT, and 25 ng tRNA in a total volume of 10 μl. The reaction mixtures were incubated at 37°C for 30 min to allow protein–RNA complex formation. The samples were mixed with 4 μl loading dye (25% glycerol, 0.2 mg/l xylencyanol and bromphenol blue) immediately before loading and separated on 4% polyacrylamide gels using Tris-borate buffer. The radioactively labeled bands were visualized with a PhosphorImager (Molecular Dynamics) and quantified with ImageQuant software 5.2.
10
Ribosome Footprints Isolation and Analysis
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The cytosol and CMX from control and treated cells were obtained as previously described and digested with micrococcal nuclease (MNase, Sigma-Aldrich, St Louis, MO) at 37°C for 30 min to obtain ribosome footprints. The reaction was stopped by the addition of 1.5 volumes of 4 M guanidine thiocyanate. Footprints were extracted by adding TRIzol and isolated by precipitating with GlycoBlue (ThermoFisher Scientific, Houston, TX) overnight.102 (link) The footprints were treated with polynucleotide kinase (ENK) at 37°C for 30 min to reverse the phosphate position. We separated 17–37 nt long footprints from the gel under UV light. We froze gel slices at −80°C and then crushed them to extract RNA.
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